The present invention relates to generation of genomic libraries in shuttle vectors and high throughput generation of vectors by homologous recombination for creating transgenic animals by homologous recombination.
A particularly productive approach to understanding the function of a particular gene in animals involves the disruption of the gene""s function by xe2x80x9ctargeted mutagenesisxe2x80x9d. A common form of targeted mutagenesis is to generate xe2x80x9cgene knockoutsxe2x80x9d. Typically, a gene knockout involves disrupting a gene in the germline of an animal at an early embryonic stage. (See, Thomas et al., Cell, 51:503 (1987).) Once established in the germline, it is possible to determine the effect of the mutation on the animal in both the heterozygous and homozygous states by appropriate breeding of mice having the germline mutation.
The mouse knockout model system is very useful for functional genomic analysis of genes. The advantages of mouse models for the study of mammalian physiology, and testing of therapies for the treatment of human diseases, and developmental abnormalities have been extensively established.
Among the many examples of the use of knockout technology utilized to investigate gene function are U.S. Pat. Nos. 5,625,122 and 5,530,178 to Mak, T. which describe the production of mice having a disrupted gene encoding lymphocyte-specific tyrosine kinase p56lck and Lyt-2, respectively. Silva et al., Science, 257:201 (1992) produced mice having a disrupted xcex1-Calcium Calmodulin kinase II gene (xcex1CaMKII gene) which resulted in animals having an abnormal fear response and aggressive behavior. (See, also, Chen et al., Science, 266:291 [1994]). Wang et al., Science, 269:1108 (1995) demonstrated that the disruption in mice of the C/EPBxcex1 gene which encodes a basic leucine zipper transcription factor results in impaired energy homeostasis in the mutant animals. Knudsen et al., Science, 270:960 (1995) demonstrated that disruption of the BAX gene in mice results in lymphoid hyperplasia and male germ cell death.
The most common approach to producing knockout animals involves the disruption of a target gene by inserting into the target gene (usually in embryonic stem cells), via homologous recombination, a DNA construct encoding a selectable marker gene flanked by DNA sequences homologous to part of the target gene. When properly designed, the DNA construct effectively integrates into and disrupts the targeted gene thereby preventing expression of an active gene product encoded by that gene.
Homologous recombination involves recombination between two genetic elements (either extrachromosomally, intrachromosomally, or between an extrachromosomal element and a chromosomal locus) via homologous DNA sequences, which results in the physical exchange of DNA between the genetic elements. Homologous recombination is not limited to mammalian cells but also occurs in bacterial cells, yeast cells, in the slime mold Dictyostelium discoideum and in other organisms. For a review of homologous recombination in mammalian cells, see Bollag et al., Ann. Rev. Genet., 23:199-225 (1989) (incorporated herein by reference). For a review of homologous recombination in fungal cells, see Orr-Weaver et al., Microbiol. Reviews, 49:33-58 (1985) incorporated herein by reference.
With the increasing awareness that animal, and particularly mouse mutations can provide such useful insights about the function of genes from humans, a great deal of interest is developing to systematically generate mutations within genes in mice that correspond to those genes which are being isolated and characterized as part of various genome initiatives such as the Human Genome Project. The problem with utilizing these procedures for large-scale mutagenesis experiments is that the technologies for generating transgenic animals and targeted mutations are currently very tedious, expensive, and labor intensive. The most tedious parts of making an animal knockout construct from a given cDNA is obtaining an appropriate genomic fragment and gene mapping. Once the genomic fragment is obtained and mapped, actual assembly of the targeting vector also is a tedious process depending upon availability of appropriate restriction sites.
Generally, the preparation of these constructs requires isolating genomic clones containing the region of interest, developing restriction maps, engineering restriction sites into the clones, and restriction digesting and ligating fragments to engineer the specific construct needed to produce the knockout. See, e.g., Mak, T. U.S. Pat. Nos. 5,625,122 and 5,530,178; Joyner et al., Nature, 338:153-156 (1989); Thomas et al., supra; Silva et al., supra, Chen et al., supra; Wang et al., supra; and Knudsen et al., supra. This is a long and tedious process that can take several months to complete. Thus, in order to more rapidly and efficiently create model organisms with genomic modifications, there exists a need to develop high throughput methods for the production of targeting constructs which do not require identification of target genomic fragments by traditional means, their cloning, and subsequent restriction mapping and other complex molecular engineering steps.
The present invention, in preferred embodiments, provides methods of preparing a genomic library for use in producing knockout targeting vectors comprising preparing a size selected mouse genomic DNA; preparing a shuttle vector comprising inserting said genomic DNA into a yeast vector, wherein the vector comprises a first bacterial origin of replication; a first bacterial selection marker; a first yeast origin of replication; a first yeast selection marker; and a first mammalian selection marker; transforming bacterial host cells with said shuttle vector to amplify said genomic library; arraying said transformed host cells into pools of cloned cells comprising shuttle vectors comprising a genomic DNA fragment; a first yeast origin of replication; a first yeast selection marker; a first bacterial origin of replication; a first bacterial selection marker; and a first selection marker for integration into mammalian cells; wherein the cells in said pools comprise mouse genomic fragments of different size.
In specific embodiments, the genomic DNA is a library which comprises mouse genomic DNA fragments ranging from about 8 kb to about 14 kb.
More particularly, the mouse genomic DNA fragments are isolated from a mouse strain selected from the group consisting of 129svj, 129 Ola, 129sv, and C57BL/6. Of course, these are merely exemplary strains of mice and those of skill in the art will be aware that other mouse strains may be employed for generating the transgenic animals of the present invention. Likewise, while certain preferred embodiments are directed to the generation of transgenic mice, it should be understood that the present invention is equally applicable to generating transgenic animals of other species such as, for example, mammals including but not limited to rabbits, mice, rats, hamsters, goats, sheep, pigs, horses, cows, dogs, cats, as well as primates, such as, monkeys, apes, and baboons.
In specific embodiments, the genomic library, when transformed into the bacterial host cells with said shuttle vector generates between about 3xc3x97106 and 5xc3x97106 clones. This is an exemplary range and it is contemplated that those of skill in the art may prepare a genomic library that generates more or fewer clones. Thus the practice of the invention may generate about 1xc3x97106 clones, about 2xc3x97106 clones, about 3xc3x97106 clones, about 4xc3x97106 clones, about 5xc3x97106 clones, about 6xc3x97106 clones, about 7xc3x97106 clones, about 8xc3x97106 clones, about 9xc3x97106 clones, about 10xc3x97106 clones or more clones or indeed may generate less than 1xc3x97106 clones and still provide meaningful shuttle vectors that may be used in the context of the present invention. The host cells that are available for transformation can be any host cell well known to those of skill in the art. In preferred embodiments, the host cells are bacterial cells selected from the group consisting of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhimurium and Serratia marcescans. Particularly preferred host cells are E. coli. 
In preferred embodiments, the shuttle vector comprises a bacterial origin of replication selected from the group consisting of ColE1-ORI, F and R1. Preferably, the bacterial origin of replication is an E. coli origin of replication. In specific embodiments, the E. coli origin of replication is ColE1-ORI. The yeast origin of replication preferred in the context of the preferred vectors of the present invention is selected from the group consisting of Cen, 2xcexcand the autonomous replication sequence.
The shuttle vectors may employ any selectable marker commonly used to monitor bacterial propagation, yeast propagation and selection in mammalian cells. In preferred embodiments, the marker for bacterial propagation is selected from the group consisting of ampicillin resistance, tetracycline resistance, neomycin resistance, kanamycin resistance and chloramphenicol resistance. These are merely exemplary and additional markers will be well known to those of skill in the art and are contemplated to be useful in the present invention. In preferred embodiments, the bacterial propagation marker for ampicillin resistance is BlaI.
Those of skill in the art will understand that any yeast selectable marker may be advantageously employed in the shuttle vectors of the present invention. In preferred embodiments, the marker for propagation in yeast is selected from the group consisting of trp1, His, Ura3, Arg, Ade and Leu2. The selectable marker for mammalian cells may be selected from the group consisting of neomycin resistance, hygromycin resistance, zeocin resistance, Salmonella HisD and puromycin N-acetyl transferase. In certain embodiments, the vectors may further comprise a negative selectable marker. In specific embodiments, the negative selectable marker is selected from the group consisting of thymidine kinase, and xanthine-guanine-phosphoribosyltransferase.
In particularly preferred embodiments, the yeast vector of the present invention comprises a BamHI site for inserting said genomic fragments. More particular embodiments contemplate that the BamHI site is flanked by priming sequences to facilitate PCR amplification. Generally, priming sequences for PCR amplification are well known to those of skill in the art; preferably, the priming sequences arc Sp6 and T7 priming sequences. In particularly preferred embodiments, the yeast vector of the present invention is the vector designated as pYYL-1.
In specific aspects the shuttle vector further comprises rare cutting enzyme sites flanking the genomic fragment. More specifically, the shuttle vector comprises rare cutting enzyme sites flanking the mammalian selection marker. In preferred aspects of the present invention, the mouse genomic library described by the present invention is used for high throughput construction of knockout vectors, and more specifically mouse knockout vectors.
Another aspect of the present invention contemplates a method for the preparation of a gene targeting vector for homologous recombination comprising selecting a bacterial clone pool positive for the gene to be targeted from an array of bacterial clones comprising the mouse genomic library described above; isolating the DNA from said positive pool; preparing a second expression construct comprising a marker cassette comprising a second yeast selectable marker and a second mammalian selectable marker, wherein said marker cassette is flanked on each side by mammalian gene-specific sequences homologous for a portion of the gene to be targeted; transforming yeast cells with the second expression construct and the DNA from the positive clone; selecting the transformed yeast cells for expression of the first and second yeast selectable markers; and isolating the targeting vector produced by the recombination between the shuttle vector and the second expression construct.
In specific embodiments, the positive pools comprising the target gene are selected by PCR analysis of the pools with gene-specific PCR primers wherein amplification of the PCR products is indicative of the pool comprising the target gene of interest. PCR amplification techniques are well known to those of skill in the art and are described in for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).
In preferred embodiments, the gene-specific flanking sequences each comprises at least about 20 nucleotides. In other embodiments , the gene specific flanking sequences each comprises from about 35 to about 400 nucleotides. This is an exemplary range and it is contemplated that the gene specific flanking sequences may comprise for example, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000 or more nucleotides.
In specific embodiments, the fragment of genomic DNA comprises from about 0.5 kb to about 5 kb of DNA on each side of a site in said gene to be targeted. This is merely an exemplary range and it is contemplated that the fragment of genomic DNA may comprise about 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1.0 kb, 1.2 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2.0 kb, 2.25 kb, 2.5 kb, 2.75 kb, 3.0 kb, 3.25 kb, 3.5 kb, 3.75 kb, 4.0 kb, 4.25 kb, 4.5 kb, 4.75 kb, 5.0 kb, 5.5 kb, 6.0 kb, 6.5 kb, 7.0 kb or more of DNA on each side of a site in said gene to be targeted. In particularly preferred embodiments, the fragment of genomic DNA comprises at least about 1 kb of genomic DNA on each side of a site in said gene to be targeted. In certain embodiments, the second yeast selectable marker is selected from the group consisting of TRP1, His, Ura3, Ade, Arg and Leu2. In other embodiments, the second mammalian selectable marker is selected from the group consisting of consisting of thymidine kinase, neomycin resistance, hygromycin resistance, Salmonella HisD and puromycin N-acetyl transferase. In specific embodiments, it is contemplated that the marker cassette comprises Ura3 as the second yeast selectable marker and the neomycin resistance gene as the second mammalian selectable marker.